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. 2017 Sep 12;91(19):e00974-17.
doi: 10.1128/JVI.00974-17. Print 2017 Oct 1.

A Unique T-Cell Receptor Amino Acid Sequence Selected by Human T-Cell Lymphotropic Virus Type 1 Tax301-309-Specific Cytotoxic T Cells in HLA-A24:02-Positive Asymptomatic Carriers and Adult T-Cell Leukemia/Lymphoma Patients

Yuko Ishihara  1 , Yukie Tanaka  1   2 , Seiichiro Kobayashi  2 , Koji Kawamura  1 , Hideki Nakasone  1 , Ayumi Gomyo  1 , Jin Hayakawa  1 , Masaharu Tamaki  1 , Yu Akahoshi  1 , Naonori Harada  1 , Machiko Kusuda  1 , Kazuaki Kameda  1 , Tomotaka Ugai  1 , Hidenori Wada  1 , Kana Sakamoto  1 , Miki Sato  1 , Kiriko Terasako-Saito  1 , Misato Kikuchi  1 , Shun-Ichi Kimura  1 , Aki Tanihara  1 , Shinichi Kako  1 , Kaoru Uchimaru  3 , Yoshinobu Kanda  4
Affiliations

A Unique T-Cell Receptor Amino Acid Sequence Selected by Human T-Cell Lymphotropic Virus Type 1 Tax301-309-Specific Cytotoxic T Cells in HLA-A24:02-Positive Asymptomatic Carriers and Adult T-Cell Leukemia/Lymphoma Patients

Yuko Ishihara et al. J Virol. .

Abstract

We previously reported that the T-cell receptor (TCR) repertoire of human T-cell lymphotropic virus type 1 (HTLV-1) Tax301-309-specific CD8+ cytotoxic T cells (Tax301-309-CTLs) was highly restricted and a particular amino acid sequence motif, the PDR motif, was conserved among HLA-A*24:02-positive (HLA-A*24:02+) adult T-cell leukemia/lymphoma (ATL) patients who had undergone allogeneic hematopoietic cell transplantation (allo-HSCT). Furthermore, we found that donor-derived PDR+ CTLs selectively expanded in ATL long-term HSCT survivors with strong CTL activity against HTLV-1. On the other hand, the TCR repertoires in Tax301-309-CTLs of asymptomatic HTLV-1 carriers (ACs) remain unclear. In this study, we directly identified the DNA sequence of complementarity-determining region 3 (CDR3) of the TCR-β chain of Tax301-309-CTLs at the single-cell level and compared not only the TCR repertoires but also the frequencies and phenotypes of Tax301-309-CTLs between ACs and ATL patients. We did not observe any essential difference in the frequencies of Tax301-309-CTLs between ACs and ATL patients. In the single-cell TCR repertoire analysis of Tax301-309-CTLs, 1,458 Tax301-309-CTLs and 140 clones were identified in this cohort. Tax301-309-CTLs showed highly restricted TCR repertoires with a strongly biased usage of BV7, and PDR, the unique motif in TCR-β CDR3, was exclusively observed in all ACs and ATL patients. However, there was no correlation between PDR+ CTL frequencies and HTLV-1 proviral load (PVL). In conclusion, we have identified, for the first time, a unique amino acid sequence, PDR, as a public TCR-CDR3 motif against Tax in HLA-A*24:02+ HTLV-1-infected individuals. Further investigations are warranted to elucidate the role of the PDR+ CTL response in the progression from carrier state to ATL.IMPORTANCE ATL is an aggressive T-cell malignancy caused by HTLV-1 infection. The HTLV-1 regulatory protein Tax aggressively promotes the proliferation of HTLV-1-infected lymphocytes and is also a major target antigen for CD8+ CTLs. In our previous evaluation of Tax301-309-CTLs, we found that a unique amino acid sequence motif, PDR, in CDR3 of the TCR-β chain of Tax301-309-CTLs was conserved among ATL patients after allo-HSCT. Furthermore, the PDR+ Tax301-309-CTL clones selectively expanded and showed strong cytotoxic activities against HTLV-1. On the other hand, it remains unclear how Tax301-309-CTL repertoire exists in ACs. In this study, we comprehensively compared Tax-specific TCR repertoires at the single-cell level between ACs and ATL patients. Tax301-309-CTLs showed highly restricted TCR repertoires with a strongly biased usage of BV7, and PDR, the unique motif in TCR-β CDR3, was conserved in all ACs and ATL patients, regardless of clinical subtype in HTLV-1 infection.

Keywords: HTLV-1 Tax; T-cell receptor; cytotoxic T cells; single-cell repertoire analysis.

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Figures

FIG 1
FIG 1
Proviral load (PVL) and profiles of CADM1 versus CD7 in CD4+ cells from ACs and ATL patients. (a) PVL of ACs (n = 17) and ATL patients (n = 3). ACs were divided into stable ACs (sACs) and high-risk ACs (hrACs) on the basis of a high PVL (more than four copies/100 PBMCs) (22). According to this definition, three ACs (AC11, AC16, and AC17) were defined as hrACs. (b) Representative profiles of CADM1 versus CD7 in CD4+ cells (HAS flow profiles) in each case of sAC, hrAC, cATL, and aATL. P (CADM1− CD7+), D (CADM1+ CD7dim), and N (CADM1+ CD7−) subpopulations were gated according to our previous report (20). (c) The proportion of CADM1+ (D + N gates) in CD4+ cells in the samples from sACs, hrACs, and ATL patients are summarized. sACs with PVLs of <4 copies/100 PBMCs had CADM1+ (D + N) values of less than 10%. hrACs (AC11, -16, and -17) with high PVLs (>4 copies/100 PBMCs) had CADM1+ values of more than 10%. Both chronic- and acute-type ATL patients had remarkably high frequencies of CADM1+ subpopulations.
FIG 2
FIG 2
Frequencies and effector memory phenotypes of Tax301-309-CTLs in ACs and ATL patients. (a) Frequencies of Tax tetramer-positive CD8 T cells among sACs (n = 14), hrACs (n = 3), and ATL patients. Horizontal bars indicate the averages of Tax tetramer-positive cells for all groups. n.s., not significant. (b) Differentiation of subsets based on CD45RA CCR7 expression in Tax tetramer-positive cells. Tax tetramer-positive cells in the CD45RA− CCR7− effector memory subset (TEM-Tax-CTL) were further evaluated according to the expression of CD27 and CD28 (24) (c) and CD27 and CD57 (25, 26) (d) and compared to that of all of the CD8 T cells in the effector memory subset.
FIG 3
FIG 3
TCR repertoire bias for Tax301-309-CTLs in ACs and ATL patients analyzed at the single-cell level. Individual Tax-tetramer-positive cells or clones detected in sACs (n = 7,782 cells and 56 clones), hrACs (n = 3,325 cells and 28 clones), and ATL patients (n = 4,351 cells and 56 clones) were analyzed with respect to the usage frequencies of TCR-β genes. The usage frequency for a given BV gene family or BJ genes is described in Results. (a and b) TCR-BV repertoires (a) and TCR-BJ repertoires (b) in Tax301-309-CTLs at the cellular and clonal levels. (c) TCR-BJ repertoires of 35 PDR motif-expressing BV7-9+Tax301-309-CTL clones from ACs and ATL patients. Black and gray bars represent TCR-BJ genes with usage frequencies above and below the mean values + 2 SDs, respectively.
FIG 4
FIG 4
Consistent observation of PDR+ clonotypes in both ACs and ATL patients and its relation to PVL. Tax-specific TCR repertoires detected in sACs, hrACs, and ATL patients were classified into four types based on the CDR3β amino acid sequences at positions 108 to 110: (i) PDR+, (ii) -DR+, (iii) P-R+, and (iv) minor repertoires. (a) Ratios of the each repertoire type to the total number of detected TCR repertoires in sACs, hrACs, and ATL patients. (b) Summary of the detection frequencies of the four repertoire types among sACs, hrACs, and ATL patients at the individual level. (c) Relationships between the absolute frequencies of PDR motif+ Tax-CTL (PDR+CTL) in PB and PVL in 13 individuals for whom a single-cell TCR repertoire analysis was performed.
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